The detrimental prospects of climate change, environmental damage and our dependency on diminishing fossil fuels resources is driving our society to develop methods for the manufacture of fuels and chemicals from renewable resources. Such renewable resources include crops, grasses, wood waste, bacteria, algae, and the like, and can generally be referred to as biomass. The use of renewable biomass resources has the potential to reduce our negative impact on the planet and to improve our sustainability.
Bacteria are of particular interest as a biomass resource because mankind has been experimenting with fermentation processes, such as those used to make beer, for three thousand years. Thus, large-scale fermentation culture technology is quite well developed. Additionally, the very first organism to be genetically engineered was E. coli, and with 40 years of recombinant DNA technology experience, the techniques for manipulating bacterial genomes are now quite reliable. Finally, the small size of bacterial genomes has allowed us to completely sequence a large variety of bacterial genomes, thus gaining insight into their metabolism and allowing us to readily manipulate their genomes and biochemical pathways. All of these factors make bacteria an attractive organism for the manufacture of specialty chemicals, such as dicarboxylic acids and their derivatives.
The small dicarboxylic acids that have 6 or fewer carbons are commercially significant chemicals with many uses. For example, the small diacids include 1,4-diacids, such as succinic acid, malic acid, and tartaric acid, and the 5-carbon molecule itaconic acid. Other diacids include the two carbon oxalic acid, three carbon malonic acid, five carbon glutaric acid and the 6 carbon adipic acid and there are many derivatives of such diacids as well.
As a group the small diacids have some chemical similarity and their uses in polymer production can provide specialized properties to the resin. Such versatility enables them to fit into the downstream chemical infrastructure markets easily. For example, the 1,4-diacid molecules fulfill many of the uses of the large scale chemical maleic anhydride in that they are converted to a variety of industrial chemicals (tetrahydrofuran, butyrolactone, 1,4-butanediol, 2-pyrrolidone and the succinate derivatives succindiamide, succinonitrile, diaminobutane and esters of succinate). Tartaric acid has a number of uses in the food, leather, metal and printing industries. Itaconic acid forms the starting material for production of 3-methylpyrrolidone, methyl-BDO, methyl-THF and others.
In particular, succinic acid or succinate—these terms are used interchangeably herein—has drawn considerable interest because it has been used as a precursor of many industrially important chemicals in the food, chemical and pharmaceutical industries. In fact, a report from the U.S. Department of Energy reports that succinic acid is one of 12 top chemical building blocks manufactured from biomass. Thus, the ability to make diacids in bacteria would be of significant commercial importance.
In fact, it is already possible to make succinate in a variety of bacteria and single cell eukaryotes, including Actinobacillus succinogenes (Guettler 1996), Anaerobiospirillum succiniciproducens (Samuelov 1991), Bacteroides fragilis (Isar 2007), Corynebacterium glutamicum (Okino 2005), Mannheimia succiniciproducens (Lee 2003), and Saccharomyces cerevisiae (Raab 2010).
Although the above microorganisms are useful, there is a strong preference for using E. coli in the industry due to its ease of genetic manipulation, fast growth, rate and growth on low cost culture medium. Efforts have been successful in engineering bacteria to increase their production of small diacids, such as succinate and the like. U.S. Pat. Nos. 7,569,380, 7,790,416, WO2008030995, U.S. Pat. Nos. 7,935,511, 7,927,859 and US2006073577 describe some of our efforts in that regard, and many other laboratories and companies have also had success (Cox 2006, Singh 2011).
However, bio-based succinate still faces the challenge of becoming cost competitive against petrochemical-based alternatives. In order to develop the bio-based industrial production of succinic acid, it will be important to grow the cells in a low cost medium, and the working strain optimally should be able to metabolize a wide range of low-cost sugar feedstock to produce succinic acid in good yields so that the cheapest available raw materials can be used.
Sucrose is the major component of the residuals from cane, sorghum and sugar beet processing, although glucose, xylose, arabinose and other sugars are also present. Because sucrose is generally cheaper than glucose, it would be a less expensive carbon source and the residuals could be cost effectively used to grow bacteria to make succinate and other diacids.
Sucrose is a disaccharide composed of glucose and fructose and while there are a few E. coli strains naturally able to utilize sucrose, many cannot due to lack of an invertase to convert the sucrose to glucose and fructose. There are a few examples where sucrose has been used by E. coli to form a compound of industrial interest, however, the few reports to use sucrose also report slow fermentation or low yields of the desired product (Donnelly 2004; Lin 2005; Andersson 2007; Blankschien 2010).
Thus, what is needed in the art are better E. coli or other bacteria for producing feedstock chemicals that can efficiently utilize sucrose as a carbon source without sacrificing growth rates or yields. Preferably said strain would be able to use a number of low cost carbon sources and produce excellent yields of succinate and other small diacids, as discussed above.